PROJECT SUMMARY Obesity is a chronic medical condition, defined by the accumulation of excess body fat and associated with high rates of morbidity and mortality. In the United States, obesity affects more than one-third of the adult population, and even more alarming, it affects about one-fifth of children and adolescents. Moreover, ethnic variations in obesity prevalence are evident, with rates in Hispanic children much higher than that of Caucasians. The obesity phenotype is essentially governed by energy homeostasis ? the intricate balance between energy intake, energy expenditure and energy storage. The complex interplay of genetic and environmental insults leading to obesity are likely mediated through this energy nexus. The ultimate goal of this project is to identify epigenetic marks that might contribute to energy homeostasis and cellular bioenergetics, which culminate in obesity risk. Using state-of-the-art next-generation sequencing technologies we will examine DNA methylation profiles in a targeted-design assay that includes several hundred well justified candidate genes and more than a hundred thousand representative CpG loci across the genome. This application makes use of the Viva la Familia Study, designed to assess genetic and environmental risk factors for obesity development in Hispanic children. It provides a unique collection of phenotypes, including anthropometry and body composition, diet and physical fitness, 24-hour calorimetry data to assess energy expenditure, eating behavior, and blood biochemistries. 916 Hispanic children from this study will undergo blood-based DNA methylation profiling to identify associations with phenotypes related to energy intake and expenditure, and obesity. We will examine DNA methylation correlations in three cohorts of paired blood-muscle samples to prioritize genes for functional analysis. To understand how DNA methylation changes associated with energy phenotypes and obesity might influence energy utilization at a cellular level, we will employ an iPSC-derived skeletal muscle cell system. For three highly significant energy- and/or obesity-associated CpG sites or differentially methylated regions, we will investigate the effect of CRISPR- mediated epigenetic modifications on gene expression and bioenergetics. We will investigate effects on cellular basal and maximal respiration, ATP-linked respiration, proton leak, fatty acid utilization and glycolysis. By investigating the role of DNA methylation in energy-related phenotypes and cellular bioenergetics, we hope to further elucidate the epigenetic mechanisms contributing to obesity. The epigenome is in a constant state of flux and is not only modified by external environmental stimuli but can be edited through epigenetic engineering. The identification of genes involved in energy balance and obesity can provide a basis for the development of targeted therapies aimed at understanding gene function, and ultimately the treatment of obesity.